Table of Contents Author Guidelines Submit a Manuscript
Evidence-Based Complementary and Alternative Medicine
Volume 2016, Article ID 8674397, 12 pages
http://dx.doi.org/10.1155/2016/8674397
Research Article

Veratri Nigri Rhizoma et Radix (Veratrum nigrum L.) and Its Constituent Jervine Prevent Adipogenesis via Activation of the LKB1-AMPKα-ACC Axis In Vivo and In Vitro

1Department of Pharmacology, Graduate School, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea
2Center for Metabolic Function Regulation, Wonkwang University, 460 Iksandae-ro, Iksan, Jeonbuk 54538, Republic of Korea
3College of Korean Medicine, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea
4College of Pharmacy, Dongduk Women’s University, 60 Hwarang-ro 13-gil, Seongbuk-gu, Seoul 02748, Republic of Korea

Received 21 August 2015; Revised 30 November 2015; Accepted 14 March 2016

Academic Editor: Antonella Fioravanti

Copyright © 2016 Jinbong Park et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

This study was performed in order to investigate the antiobese effects of the ethanolic extract of Veratri Nigri Rhizoma et Radix (VN), a herb with limited usage, due to its toxicology. An HPLC analysis identified jervine as a constituent of VN. By an Oil Red O assay and a Real-Time RT-PCR assay, VN showed higher antiadipogenic effects than jervine. In high-fat diet- (HFD-) induced obese C57BL/6J mice, VN administration suppressed body weight gain. The levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT-enhancer-binding protein alpha (C/EBPα), adipocyte fatty-acid-binding protein (aP2), adiponectin, resistin, and LIPIN1 were suppressed by VN, while SIRT1 was upregulated. Furthermore, VN activated phosphorylation of the liver kinase B1- (LKB1-) AMP-activated protein kinase alpha- (AMPKα-) acetyl CoA carboxylase (ACC) axis. Further investigation of cotreatment of VN with the AMPK agonist AICAR or AMPK inhibitor Compound C showed that VN can activate the phosphorylation of AMPKα in compensation to the inhibition of Compound C. In conclusion, VN shows antiobesity effects in HFD-induced obese C57BL/6J mice. In 3T3-L1 adipocytes, VN has antiadipogenic features, which is due to activating the LKB1-AMPKα-ACC axis. These results suggest that VN has a potential benefit in preventing obesity.

1. Introduction

Obesity has become a public health dilemma recently, especially in developed countries. According to the report of the World Health Organization, over 1.4 billion of 20-year-old or older individuals worldwide are overweight [1]. Obesity is closely related to chronic diseases such as hyperlipidemia, hypertension, and type 2 diabetes mellitus [2]. Adipogenesis is a process by which undifferentiated preadipocytes are converted into fully differentiated adipocytes, such as fat cells [3]. The mouse preadipocyte cell line 3T3-L1 is one of the best characterized models for studying the conversion process of preadipocytes into adipocytes. Adipogenesis is known as a closely related process to the etiologies of obesity involving several genes and proteins at different stages [4].

During the adipogenesis of 3T3-L1 cells, among the several adipogenic transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT-enhancer-binding protein alpha (C/EBPα) are known to act as the key regulators [5]. PPARγ acts as a regulator of development of adipocytes and is known as the only factor that can induce the adipocyte-like phenotype in nonadipogenic cell types [6]. C/EBPα, a member of the leucine zipper transcription factor family, plays an important role in the terminal differentiation in adipocytes [7]. These two factors are not expressed in preadipocytes but are activated during adipocyte differentiation [8]. Adipocyte fatty-acid-binding protein (aP2) acts as cytoplasmic lipid chaperones and plays a role in several lipid signals [9], while resistin, a newly identified adipokine, is secreted by adipocytes and has antagonistic effects on insulin actions [10]. A novel protein, LIPIN1, is primarily expressed in adipose tissues, liver, and skeletal muscles [11]. SIRT1 has been found to suppress adipocyte differentiation and to prevent TG accumulation in white adipose tissue through repression of PPARγ [12]. Similarly, AMP-activated protein kinase (AMPK) activation inhibits adipocyte differentiation and lipogenesis [13, 14].

The fuel-sensing enzyme AMPK is a heterotrimeric protein kinase consisting of three subunits: α, β, and γ [15]. The increased AMP/ATP ratio affects the γ subunit to induce phosphorylation of a threonine residue within the activation domain of the α subunit, by the upstream kinase, liver kinase B1 (LKB1) [16]. AMPK can be activated by inhibition of ATP, that is, hypoxia, ischemia, oxidative stress, and glucose deprivation [15] but, importantly, can be activated by adipokines leptin and adiponectin, the important regulators of energy metabolism [17]. Activation of AMPK results in the repression of ATP-consuming anabolic processes and activation of ATP-producing catabolic processes [17, 18]. AMPK mediates these effects through the phosphorylation of metabolic enzymes, such as acetyl CoA carboxylase (ACC), the rate-limiting enzyme for fatty acid oxidation [19].

Veratrum nigrum L., commonly known as black false hellebore, is a coarse, poisonous perennial herb native to Asia and Europe [20]. The stems and roots of this plant are used under the name of Veratri Nigri Rhizoma et Radix. Due to its ability to cause nausea and vomiting, it is applied to dyspnea in epilepsy or stroke patients in Traditional Korean Medicine. Previous studies report that Veratrum nigrum L. is a potential agonist of β2-adrenoceptor [21] and is also able to prevent hepatic ischemia injury in rats [22]. However, the effect of the ethanolic extract of Veratri Nigri Rhizoma et Radix (VN) or its constituent, jervine ((3β,23β)-17,23-epoxy-3-hydroxyveratraman-11-one; Figure 1), on obesity has not been reported to date. Thus, this study was performed to investigate the effects of VN and jervine on obesity in vivo and in vitro.

Figure 1: Structure of jervine.

2. Materials and Methods

2.1. Sample Collection

The stems and roots of Veratrum nigrum L. (Veratri Nigri Rhizoma et Radix), known as “black false hellebore,” were obtained from Omniherb (Daejeon, Republic of Korea). A voucher specimen of the plant has been deposited in our laboratory. The Veratri Nigri Rhizoma et Radix was already processed into dried and chopped pieces before purchase. 100 g of Veratri Nigri Rhizoma et Radixslices was extracted for 2 h 20 min using a heating mantle with 1000 mL of 70% aqueous ethanol. The extract was filtered through a 0.22 μm syringe filter, evaporated, and then stored at −20°C until usage.

2.2. Reagents

Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin-glutamine, bovine serum (BS), and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). Insulin, 3-isobutylmethylxanthine (IBMX), dexamethasone (DEX), Oil Red O powder, and 5-amino-4-imidazolecarboxamide riboside (AICAR) were from Sigma Chemical Co. (St. Louis, MO, USA). 6-[4-(2-Piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine (Compound C) was obtained from Calbiochem (La Jolla, CA, USA). The antibodies for C/EBPα and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and PPARγ, phospho-LKB1, phospho-ACC, phospho-AMPKα, and AMPKα were obtained from Cell Signaling technology (Beverly, MA, USA). Jervine (PubChem CID: 10098) was purchased from Sigma Chemical Co. (St. Louis, MO, USA).

2.3. HPLC Analysis

The chromatographic system consisted of Jasco HPLC-LC-2000 Plus (Tokyo, Japan) equipped with a Jasco MD-2018 Plus Photodiode Array Detector, using the Mightysil RP-18(L) GP column (5 μm, 4.6 × 150 mm, Kanto Chemical Co. Inc., Japan). The column temperature was set to 40°C. The mobile phase consisted of acetonitrile as solvent A and acetic acid in water (0.05%) as solvent B using gradients elution. The initial mobile phase composition was 10% of solvent A, and the following gradient system was used: 10–20% (0–10 min), 20% (10–15 min), 20–35% (15–25 min), 35% (25–30 min), 35–40% (30–35 min), 40% (35–45 min), 40–10% (45–50 min), and 10% of solvent A (50–60 min). The total running time was 60 min, and the flow rate was 1.0 mL/min. Data acquisition was performed in the range of 190–650 nm. The retention times of these compounds were obtained as follows: jervine, 26.3 min.

2.4. Animal Experiments

The animal obesity model experiment was conducted based on previous reports [20, 2325]. Male C57BL/6J mice, weighing 17-18 g at the age of 4 weeks, were purchased from the Dae-Han Experimental Animal Center (Eumsung, Republic of Korea). The animal experiment was proceeded in conditions in accordance with the regulations issued by the Institutional Review Board of Kyung Hee University (confirmation number: KHUASP (SE)-13-012). The mice were maintained for 1 week prior to the experiments in a 12-hour light/dark cycle at humidity of 70% and constant temperature of °C. The animals were then divided into four groups ( per group): a normal control group fed normal chow diet (CJ Feed Co. Ltd., Seoul, Republic of Korea), a high-fat diet (HFD) group fed 60% fat HFD (Rodent diet D12492, Research diet, New Brunswick, NJ, USA) for 14 weeks, a VN group and slinti group which were fed HFD for 4 weeks in order to induce obesity and then fed for 10 additive weeks with HFD plus VN or HFD plus slinti (Myungmoon Pharm. Co. LTD., Seoul, Republic of Korea), respectively. Slinti, which consisted of Theae Folium Powder 250 mg and Orthosiphon Powder 150 mg, was used as a positive control according to the antiobese effects reported on our previous studies [20, 24]. The components of the diets are described in S1 Table (in Supplementary Material available online at http://dx.doi.org/10.1155/2016/8674397). The body weight and food intake amount were recorded every other day. At the end of the period of total 14 weeks, the animals were fasted overnight. The next day, they were anesthetized under CO2 asphyxiation and plasma was separated at 4,000 g for 30 min immediately after blood collection via cardiac puncture. The total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, alanine transaminase (ALT), and creatinine were assessed by Seoul Medical Science Institute (Seoul, Republic of Korea). Animals were killed by cervical dislocation. Subcutaneous white adipose tissues (sWATs) were weighed.

2.5. Cell Culture and Differentiation

3T3-L1 mouse embryo fibroblasts were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were grown in DMEM plus 10% BS containing penicillin-streptomycin-glutamine solution (100 UI/mL) in a 10 cm dish. After reaching passage 10, the cells were then moved to 6-well plates for final differentiation in DMEM plus 10% FBS containing antibiotics described above. Until 100% confluence (Day 0), the cells were maintained in a cell incubator at 37°C, 5% CO2, and 95% humidity. Two days after confluence (Day 2), the cells were stimulated to differentiation with differentiation media (DM) composed of DMEM plus 10% FBS and differentiation inducers (MDI: 1 μM DEX, 500 μM IBMX, and 1 μg/mL insulin). After 2 days, the DM was removed and replaced with DMEM plus 10% FBS containing 1 μg/mL insulin (Day 4). After another additional 2 days, the media were replaced with DMEM plus 10% FBS containing 1 μg/mL insulin again (Day 6). The cells were cultured for 2 more days, at which time more than 90% of cells were mature adipocytes with accumulated fat droplets, and then harvested for further experiments (Day 8). The VN or jervine was treated at Day 4, dissolved in the culture media. AICAR or Compound C was administered 30 min before the VN or jervine treatment dissolved in the culture media.

2.6. MTS Assay

The 3T3-L1 preadipocytes were seeded (2 × 104 cell/well) and incubated in DMEM plus 10% FBS for 24 h. Then the cells were incubated in the same media containing an ethanol extract of VN for an additional 48 h. Cell viability was monitored using the cell proliferation MTS kit by the Promega Corporation (Madison, WI, USA) as recommended by the manufacturer. Prior to measuring the viability, the media were removed and replaced with 200 μL of fresh DMEM plus 10% FBS medium and 10 μL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) solution. The cells were then incubated in the incubator for 4 h. The absorbance was measured at 490 nm in a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA) to determine the formazan concentration, which is proportional to the number of live cells.

2.7. Oil Red O Staining

Intracellular lipid accumulation was measured using Oil Red O. The Oil Red O working solution was prepared as described by Ramírez-Zacarías et al. [26]. Briefly, Oil Red O stock solution was prepared Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) dissolved in isopropanol at the concentration of 3.5 mg/mL, and the Oil Red O working solution was prepared 60% Oil Red O stock solution mixed with 40% distilled water. 3T3-L1 adipocytes were harvested 6 days after the initiation of differentiation. Cells were washed twice with phosphate buffered saline (PBS, pH 7.4) and then fixed with 10% neutral formalin for 2 hours at room temperature. After washing with 60% isopropanol, the cells were stained with Oil Red O working solution for 30 min and then were washed 4 times with water in order to remove the unbound dye. The stained cells were observed by an Olympus IX71 Research Inverted Phase microscope (Olympus Co., Tokyo, Japan). Following the microscopic observation, 100% isopropanol was added as an extraction solution to extract the staining dye of cells. The absorbance of the extracted dye was measured spectrophotometrically at 500 nm in a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA).

2.8. RNA Isolation and Real-Time RT-PCR

Total cellular RNA was isolated from 3T3-L1 adipocytes using QIAzol lysis reagent (Qiagen Sciences, Maryland, USA). Total RNA was used as a template for first-strand cDNA synthesis performed using a Power cDNA Synthesis Kit (iNtRON Biotechnology, Seoul, Korea) according to the manufacturer’s instructions. The Real-Time RT-PCR mixture, with a final volume of 20 μL, consisted of Fast SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 1 μM of a forward primer, 1 μM of a reverse primer, and 0.1 μg of a cDNA sample. The thermal cycling conditions were as follows: holding stage, 10 s at 95°C and 40 cycles of 15 s at 95°C and 1 min at 60°C, and then melt curve stage, 15 s at 95°C, 1 min at 60°C, and 15 s at 95°C. PCR products were measured with a StepOnePlus Real-Time RT-PCR System (Applied Biosystems, Foster City, CA, USA), and the relative gene expression was calculated based on the comparative CT method using a StepOne Software v2.1 (Applied Biosystems, Foster City, CA, USA). The mRNA expression of GAPDH was used as an endogenous control. The target cDNA was amplified using the sense and antisense primers described in S2 Table.

2.9. Western Blot Analysis

After experimental treatment, cells were washed twice with ice-cold PBS and lysed with RIPA lysis buffer, which consisted of 50 mM Tris-HCl (pH 7.5), 0.1% sodium dodecyl sulphate (SDS), 0.1% Triton X-100, 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride. Insoluble materials were removed by centrifugation at 13,000 rpm for 20 min at 4°C. The total concentration of extracted proteins was determined using the method of Bradford [27]. The proteins in the supernatants were separated by 8% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 10 mM Tris, 150 mM NaCl, and 0.05% Tween-20 (TBST) (pH 7.6) containing 5% skim milk for 1 h at room temperature, the membranes were washed with TBST and then incubated with the appropriate primary antibodies against PPARγ, C/EBPα, phospho-AMPKα, AMPKα, phospho-ACC, phospho-LKB1, or GAPDH at 4°C overnight. After washing with TBST, the blots were subsequently incubated with horseradish peroxidase- (HRP-) conjugated AffiniPure Goat anti-rabbit IgG (Jackson ImmunoResearch Lab., West Grove, PA, USA) or HRP-conjugated AffiniPure Goat anti-mouse IgG (Jackson ImmunoResearch Lab., West Grove, PA, USA) in 5% skim milk-TBST at room temperature for 1 h. Protein signals were developed by using the ECL Western Blotting Detection Reagent (Amersham Bioscience, Piscataway, NJ, USA). All experiments were repeated at least three times. PVDF membranes were purchased from Millipore (EMD Millipore Co., Billerica, MA, USA), and the protein assay reagents were obtained from Bio-Rad (Bio-Rad Laboratories, Hercules, CA, USA).

2.10. Statistical Analysis

Data were expressed as the mean ± standard deviation (SD). Significant differences between groups were determined using Student’s t-test and one-way ANOVA followed by post hoc Tukey’s multiple comparisons tests. All statistical analyses were performed using SPSS statistical analysis software version 11.5 (SPSS Inc., Chicago, IL, USA). A probability value of was considered as statistical significance.

3. Results

3.1. HPLC Analysis of VN

For qualitative analysis of VN to confirm jervine, we performed an HPLC analysis. HPLC-PDA measurement of the ethanolic extraction of VN demonstrated various chromatographic peaks. Comparing the analyzed chromatographic peaks with reference chromatographic peaks, jervine was identified (Figure 2(a)).

Figure 2: HPLC analysis of VN and effects of VN and its compound jervine on 3T3-L1 adipocytes. (a) HPLC-PDA measurement of VN demonstrated various chromatographic peaks. By comparing chromatographic peaks with reference chromatographic peaks, jervine was identified. (b) The effects of VN and jervine on lipid accumulation during 3T3-L1 adipogenesis were compared by an Oil Red O staining assay. (c) The effects of VN and jervine on adipogenic genes, PPARγ and C/EBPα, expressions were compared using a Real-Time RT-PCR assay. Data are expressed as mean ± SD of three or more experiments. versus MDI-uninduced preadipocytes, , and versus MDI-induced adipocytes.
3.2. Comparisons of Antiadipogenic Effects of VN and Its Compound Jervine

As the HPLC analysis identified jervine as a compound of VN, investigation to compare the two substances was proceeded. Ahead of any further in vitro experiments, a cell viability test was performed. As a result, VN did not show any cytotoxicity at the concentration of 0.01–1 mg/mL (Supplementary Figure (a)). Due to this result, further assays were performed at concentrations of 0.01, 0.1, and 1 mg/mL. The MTS assay also showed that jervine had no cytotoxicity at the concentration up to 10 μM (Supplementary Figure (b)). In order to compare the effects of VN and jervine on lipid accumulation, an Oil Red O assay was performed. The assay result showed that 1 mg/mL of VN had a slightly higher inhibition rate (23.46%) on lipid accumulation than 10 μM of jervine (21.31%), but there were no significant differences between the two (Figure 2(b)). A Real-Time RT-PCR assay was performed to investigate the effects on adipogenic genes PPARγ and C/EBPα. As in Figure 2(c), VN had higher inhibition rate on both genes and, especially on C/EBPα expression, it showed a significantly higher inhibition rate (53.69%) compared to jervine (28.17%). These results suggest that VN and jervine both have antiobese features, while VN might have higher effects than its compound jervine. Therefore, further experiments were performed in order to investigate the effects of VN, not jervine.

3.3. VN Has Beneficial Effects on HFD-Induced Obese C57BL/6J Mice

To investigate the antiobesity effects of VN in vivo, an animal experiment was performed as described in the materials and methods section. As shown in Figure 3(a), VN treatment significantly suppressed body weight gain ( g) when compared to the HFD group ( g), which was even greater than the positive control, slinti group ( g). Furthermore, the weight of the sWATs between the VN group and HFD group showed significant difference (Figure 3(b)). The blood serum analysis revealed the beneficial effects of VN on total cholesterol, triglyceride, and LDL-cholesterol levels (Figures 3(c), 3(d), and 3(e)). In particular, the serum LDL-cholesterol level of the VN group was highly downregulated compared to that of the HFD group. In addition, in spite of the concern on toxic features of VN, the VN group did not show any toxicity in the liver and kidney as proved by the serum ALT and creatinine levels (Supplementary Figure ).

Figure 3: Effect of VN in HFD-induced obese mice. (a) The body weight changes of the NC group, HFD group, HFD + VN group, and HFD + slinti group were measured every week. (b) The subcutaneous adipose tissue weights were measured after the termination of the experiment. The serum levels of (c) total cholesterol, (d) triglyceride, (e) LDL-cholesterol, and (f) HDL-cholesterol were measured. Data are expressed as mean ± SD (). versus NC group, , and versus HFD-induced obese group.
3.4. VN Inhibits Lipid Accumulation in 3T3-L1 Adipocytes

Next, to investigate the effects of VN on adipocyte differentiation, the lipid accumulation was measured using the Oil Red O staining method. As in Figures 4(a) and 4(b), VN significantly suppressed lipid accumulation at the dose of 0.1 and 1 mg/mL, suggesting its antiadipogenic effect. Epigallocatechin-3-gallate (EGCG), a green tea compound previously reported to show antiobese features [28], was used as a positive control.

Figure 4: Effect of VN on lipid accumulation during 3T3-L1 adipocyte differentiation. (a) The lipid droplets were observed at the magnification of 100x. (b) The lipid content was quantified by resolving the Oil Red O stain in isopropanol and measuring absorbance at 500 nm. EGCG was used as a positive control. Data are expressed as mean ± SD of three or more experiments. versus MDI-uninduced preadipocytes, , and versus MDI-induced adipocytes.
3.5. VN Modulates Adipogenic Gene Expressions in 3T3-L1 Adipocytes

Among the several related factors in adipogenesis, PPARγ and C/EBPα especially are well known as the two major regulators in managing adipogenesis [29]. Figure 5(a) shows that VN treatment suppressed PPARγ and C/EBPα gene expression at 0.1 and 1 mg/mL. Further investigations on protein levels were performed in order to confirm the antiadipogenic effects of VN. As shown in Figure 5(b), VN treatment successfully downregulated the protein levels of PPARγ and C/EBPα.

Figure 5: Effect of VN on adipogenesis-related factors in 3T3-L1 adipocytes. The mRNA expression levels of (a) PPARγ and C/EBPα, (c) aP2, adiponectin, and resistin, and (d) LIPIN1 were measured by the Real-Time RT-PCR assays. (b) The expressions PPARγ and C/EBPα were measured using a Western blot assay. GAPDH was used as an endogenous control. EGCG was used as a positive control. Data are expressed as mean ± SD of three or more experiments. versus MDI-uninduced preadipocytes, , and versus MDI-induced adipocytes.

We also examined the effects of VN on adipogenic genes aP2, resistin, and adiponectin. The downstream target genes of PPARγ and C/EBPα, such as aP2 and adiponectin, are involved in maintaining the adipocyte phenotype [30], and resistin has been reported as a link between obesity and diabetes [10]. These three adipokines were also downregulated by VN treatment, at a dose-dependent manner (Figure 5(c)).

LIPIN1 is an adipokine which has an important role in the regulation of cellular lipid and energy metabolism [31]. As in Figure 5(d), LIPIN1 was suppressed by VN at a dose-dependent manner.

These results suggest the beneficial effects of VN on obesity, as it suppresses adipogenic factors expressed during the differentiation of 3T3-L1 adipocytes, at both the mRNA and protein levels.

3.6. VN Activates Phosphorylation of the LKB1-AMPKα-ACC Axis in 3T3-L1 Adipocytes

Next, we investigated whether VN can influence the SIRT1-AMPK axis. SIRT1, one of the seven mammalian orthologs (SIRT1–SIRT7), is a conserved NAD+-dependent protein deacetylase [32], which is known to suppress adipogenesis [12]. SIRT1 was upregulated by the VN treatment, but only at the highest concentration of 1 mg/mL (Figure 6(a)). As SIRT1 is a closely related factor to AMPKα in obesity, we assessed the effects of VN on AMPKα and its upstream and downstream targets, LKB1 and ACC.

Figure 6: Effect of VN on AMPKα pathway-related factors in 3T3-L1 adipocytes. (a) The mRNA expression level of SIRT1 was measured by the Real-Time RT-PCR assay. (b) The expressions of p-AMPKα, p-ACCα, and p-LKB1 were measured using a Western blot assay. (c) The effects of VN on the AMPKα modulation when administered with AMPK activator AICAR or AMPK inhibitor Compound C were evaluated by a Western blot assay. AMPKα was used as an endogenous control for p-AMPKα measurement. GAPDH was used as an endogenous control. EGCG was used as a positive control. Data are expressed as mean ± SD. of three or more experiments. versus MDI-uninduced preadipocytes, , and versus MDI-induced adipocytes.

As our hypothesis, VN treatment could induce the phosphorylation of AMPKα (Figure 6(b)). AMPKα is a key player in energy homeostasis, and its activation results in inhibition of adipocyte differentiation [13] and lipogenesis via increased ACC phosphorylation [14]. AMPKα phosphorylation was successfully activated in the VN treated cells. However, interestingly, VN treatment failed to activate phosphorylation of both the AMPK upstream kinase LKB1 and the AMPK downstream target ACC (Figure 6(b)). The Western blot results suggested that VN treatment activates phosphorylation of AMPK directly, without affecting the phosphorylation levels of LKB1 or ACC.

AICAR is an AMPK agonist and, in contrast, Compound C acts as an inhibitor of AMPK. Sullivan and colleagues reported that AICAR was able to activate AMPK in a time- and dose-dependent manner and therefore inhibit lipogenesis [14]. On the other hand, Compound C, also known in the name dorsomorphin, is reported to be the only available agent that is used as a cell-permeable AMPK inhibitor [33]. As in Figure 6(c), AICAR treatment upregulated AMPKα phosphorylation while Compound C was able to suppress the phosphorylation of AMPKα. VN treatment could not boost up the effect of AICAR but, on the other hand, it was able to show compensation to the AMPKα inhibition of Compound C and highly upregulated the phosphorylation of AMPKα. These results confirm the antiadipogenic effects of VN, supposably by its ability to activate AMPKα phosphorylation.

4. Discussion

In this study, we have evaluated the effects of VN and its constituent jervine on obesity using the in vivo HFD-induced obese mouse model and the in vitro 3T3-L1 adipocyte model, for the first time.

Obesity is a chronic metabolic disorder caused by an imbalanced energy intake-expenditure status [34]. The prevalence of obesity is growing; in the year 2008, the worldwide obesity has nearly doubled since 1980 [1]. Current medications for the treatment of obesity include mixed noradrenergic-serotonergic agents (sibutramine) [35] and absorption-reducing agents (orlistat) [36]. However, these two drugs show adverse effects at high frequencies. For example, sibutramine is reported to cause cardiac arrhythmias, constipation, and headache with only minimum weight loss [35], and orlistat can show steatorrhea and lipid-soluble-vitamin-deficiency [36]. Due to the limits of currently available drugs, the necessity for new drugs for the treatment of obesity is rapidly growing, and the interest in natural products especially is increasing.

Veratrum nigrum L. is a medicinal plant used in Traditional Chinese and Korean Medicine native to Asia and Europe. In the plant, mainly the stem and root of Veratrum nigrum L., Veratri Nigri Rhizoma et Radix, are administered internally as an emetic medicine in cases of strokes or epilepsies or also topically treated in order to kill parasites or to stop pruritus [37]. But because of its toxicology, Veratri Nigri Rhizoma et Radix is not widely used, as it is difficult to prepare a safe yet effective dose [38]. Therefore, only few reports on Veratri Nigri Rhizoma et Radix are currently published. Among those studies, none has reported the effects of VN on obesity or adipogenesis.

Jervine (C27H39O3N), a steroidal alkaloid derived from the Veratrum genus [39], which is reported to have antitumor effects [40, 41], was detected as an active compound of VN by the HPLC analysis. Jervine and VN both successfully suppressed lipid accumulation and expressions of adipogenic genes PPARγ and C/EBPα in 3T3-L1 adipocytes. However, the antiadipogenic effects of VN were higher than jervine, and thus further investigations were performed in order to assess the effects of VN.

As the basic in vitro experiments preceded suggested positive effects on obesity, an in vivo experiment was carried on using C57BL/6J mice. As expected, VN had beneficial effects on obesity in the animal model, too. The weight gains and sWAT weights were significantly suppressed in the VN administered group. Serum analyses also confirmed the beneficial effects of VN on obesity. On the other hand, ALT and creatinine, the barometers measuring liver and kidney toxicity, respectively, were not negatively affected but showed lower levels than the HFD group. These results are conflict to the formerly known toxicity of Veratrum nigrum [38, 42]. The in vivo results do not only prove the beneficial effects of VN in obesity, but the toxicity-safe dosage of VN also shows potential application to human treatment as well, leading to expansion of the limited oral use of VN.

Based on the positive in vivo results on obesity, we then performed more experiments back at the cell level, in order to find out which responsible mechanism was giving the beneficial effects. First, an Oil Red O staining assay showed suppressed lipid accumulation by VN treatment. In addition, the mRNA levels of adipogenic genes including PPARγ, C/EBPα, aP2, adiponectin, resistin, and LIPIN1 were downregulated. The suppression of the genes suggested the inhibiting effect on adipogenesis by VN treatment. PPARγ and C/EBPα are well known as important regulators of adipogenesis [57], while adipose-derived adipokines, aP2, adiponectin, and resistin possess their roles in adipocytes in lipid signaling [9], glucose regulation [43], and insulin resistance [10], respectively. On the other hand, LIPIN1 is a candidate gene for lipodystrophy [11]. In addition to these genes, the level of SIRT1, the NAD+-dependent protein deacetylase [12], which is able to suppress adipogenesis, was significantly upregulated by VN at the highest dose of 1 mg/mL. The elevated SIRT1 expression suggested the effects of VN on the SIRT1-AMPKα axis, which is a key factor in the etiology of obesity.

Previous studies have reported the detailed role of SIRT1-AMPKα axis in obesity. According to Ruderman et al., AMPKα is suggested to play a central role in metabolic syndromes [44]. Other numerous studies also link the phosphorylation of AMPK to obesity in 3T3-L1 models [3, 8, 24, 45, 46]. Several genetic rodent models with a metabolic syndrome phenotype, such as ob/ob mice or fa/fa rats, show decreased AMPK activity [47], and when the decreased AMPK activity is restored by AICAR, they showed improved glucose homeostasis [48, 49]. Sirtuins, a group of histone/protein deacetylases, are regulated by the NAD+/NADH ratio. SIRT1 is the most well-known member of this family, which is reported to respond to changes in energy expenditure [47], which is similar to AMPK. Other studies revealed that SIRT1 can activate AMPK by deacetylating LKB1, the upstream kinase of AMPK [50, 51], and vice versa AMPK can activate SIRT1 by increasing the NAD+/NADH ratio [52]. Therefore, these previous reports suggest the important role of SIRT1-AMPKα axis, or circle, in obesity.

The Real-Time RT-PCR result showing the upregulation of the antiadipogenic gene SIRT1 by VN treatment suggested the possible effects of VN on the SIRT1-AMPKα circle. As we expected, a Western blot analysis confirmed the effect of VN on AMPKα phosphorylation, subsequently to the previous results. VN treatment also suppressed the expression of PPARγ and C/EBPα at the protein levels. Unlike AMPKα, however, the phosphorylation of ACC and LKB1, the upstream and downstream enzymes of AMPKα, respectively, were not upregulated as we expected. These results were conflict to our former researches, in which the protein expressions of p-ACC or p-LKB1 were successfully elevated by treatments showing antiobese features [8, 24]. LKB1, also known as serine/threonine kinase 11, is a protein kinase encoded from the LKB1 gene [53]. Originally known as a tumor suppressor, LKB1 is also related to obesity due to its role as an upstream factor of the energy homeostasis regulator, AMPK [54]. The downstream target of AMPK, ACC, is dephosphorylated by AMPK inhibition [55], and activation of AMPK leads to inhibition of cholesterol synthesis by direct phosphorylation of ACC [56]. However, recent studies report that p-LKB1 [57] or p-ACC [58] is not essential in the cascade of AMPK phosphorylation.

In contrast to the p-ACC and p-LKB1 expressions, the effect of VN on AMPKα activation was surely confirmed, as we coadministered the AMPK activator AICAR and the AMPK inhibitor Compound C with VN. AICAR and Compound C were able to activate or attenuate the phosphorylation of AMPKα. In addition, the treatment of VN was able to restore the inhibited AMPKα phosphorylation by Compound C, nearly up to the AMPK level by AICAR activation. These cotreatment results suggest the effect of VN on the LKB1-AMPKα-ACC axis, by solely affecting the AMPK activation only.

Our results on sole phosphorylation of AMPK in the LKB1-AMPK-ACC pathway suggest that the antiadipogenic features of VN resulted from direct activation of AMPK by VN and proceed through SIRT1 activation, which leads to inhibition of PPARγ and C/EBPα. However, the detailed mechanism for how VN regulates adipogenesis regarding sole activation of AMPK within the LKB1-AMPK-ACC axis requires further investigation.

5. Conclusions

In summary, our results demonstrated that VN contains jervine, and they both can attenuate lipid accumulation during 3T3-L1 adipogenesis. VN showed beneficial effects on obesity in a HFD-induced obese C57BL/6J mouse model. In 3T3-L1 adipocytes, VN was able to attenuate adipogenic factors and upregulate SIRT1 and AMPKα phosphorylation, suggesting its ability to activate the SIRT1-AMPKα circle. These results led to further investigations involving the LKB1-AMPKα-ACC axis. VN treatment was able to compensate for the action of the AMPK inhibitor, Compound C. These results suggest the potential of VN as an AMPKα axis-modulating antiobese agent.

Competing Interests

The authors declare that they have no competing interests.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2011-0012113).

References

  1. WHO, “WHO Media Centre Fact Sheets: Obesity and Overweight,” 2015, http://www.who.int/mediacentre/factsheets/fs311/en/.
  2. P. G. Kopelman, “Obesity as a medical problem,” Nature, vol. 404, no. 6778, pp. 635–643, 2000. View at Google Scholar · View at Scopus
  3. H.-L. Kim, J.-E. Sim, H.-M. Choi et al., “The AMPK pathway mediates an anti-adipogenic effect of fruits of Hovenia dulcis Thunb,” Food and Function, vol. 5, no. 11, pp. 2961–2968, 2014. View at Publisher · View at Google Scholar · View at Scopus
  4. J. M. Ntambi and Y.-C. Kim, “Adipocyte differentiation and gene expression,” The Journal of Nutrition, vol. 130, no. 12, pp. 3122S–3126S, 2000. View at Google Scholar · View at Scopus
  5. Z. Wu, E. D. Rosen, R. Brun et al., “Cross-regulation of C/EBPα and PPARγ controls the transcriptional pathway of adipogenesis and insulin sensitivity,” Molecular Cell, vol. 3, no. 2, pp. 151–158, 1999. View at Publisher · View at Google Scholar · View at Scopus
  6. P. Tontonoz and B. M. Spiegelman, “Fat and beyond: the diverse biology of PPARγ,” Annual Review of Biochemistry, vol. 77, pp. 289–312, 2008. View at Publisher · View at Google Scholar · View at Scopus
  7. R. P. Brun, J. B. Kim, E. Hu, S. Altiok, and B. M. Spiegelman, “Adipocyte differentiation: a transcriptional regulatory cascade,” Current Opinion in Cell Biology, vol. 8, no. 6, pp. 826–832, 1996. View at Publisher · View at Google Scholar · View at Scopus
  8. M.-Y. Jeong, H.-L. Kim, J. Park et al., “Rubi fructus (Rubus coreanus) inhibits differentiation to adipocytes in 3T3-L1 cells,” Evidence-Based Complementary and Alternative Medicine, vol. 2013, Article ID 475386, 10 pages, 2013. View at Publisher · View at Google Scholar · View at Scopus
  9. G. Tuncman, E. Erbay, X. Hom et al., “A genetic variant at the fatty acid-binding protein aP2 locus reduces the risk for hypertriglyceridemia, type 2 diabetes, and cardiovascular disease,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 18, pp. 6970–6975, 2006. View at Publisher · View at Google Scholar · View at Scopus
  10. C. M. Steppan, S. T. Bailey, S. Bhat et al., “The hormone resistin links obesity to diabetes,” Nature, vol. 409, no. 6818, pp. 307–312, 2001. View at Publisher · View at Google Scholar · View at Scopus
  11. M. Péterfy, J. Phan, P. Xu, and K. Reue, “Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin,” Nature Genetics, vol. 27, no. 1, pp. 121–124, 2001. View at Publisher · View at Google Scholar · View at Scopus
  12. F. Picard, M. Kurtev, N. Chung et al., “Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-γ,” Nature, vol. 429, no. 6993, pp. 771–776, 2004. View at Publisher · View at Google Scholar · View at Scopus
  13. S. A. Habinowski and L. A. Witters, “The effects of AICAR on adipocyte differentiation of 3T3-L1 cells,” Biochemical and Biophysical Research Communications, vol. 286, no. 5, pp. 852–856, 2001. View at Publisher · View at Google Scholar · View at Scopus
  14. J. E. Sullivan, K. J. Brocklehurst, A. E. Marley, F. Carey, D. Carling, and R. K. Beri, “Inhibition of lipolysis and lipogenesis in isolated rat adipocytes with AICAR, a cell-permeable activator of AMP-activated protein kinase,” FEBS Letters, vol. 353, no. 1, pp. 33–36, 1994. View at Publisher · View at Google Scholar · View at Scopus
  15. Y. C. Long and J. R. Zierath, “AMP-activated protein kinase signaling in metabolic regulation,” The Journal of Clinical Investigation, vol. 116, no. 7, pp. 1776–1783, 2006. View at Publisher · View at Google Scholar · View at Scopus
  16. R. J. Shaw, M. Kosmatka, N. Bardeesy et al., “The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 10, pp. 3329–3335, 2004. View at Publisher · View at Google Scholar · View at Scopus
  17. M. C. Towler and D. G. Hardie, “AMP-activated protein kinase in metabolic control and insulin signaling,” Circulation Research, vol. 100, no. 3, pp. 328–341, 2007. View at Publisher · View at Google Scholar · View at Scopus
  18. G. R. Steinberg, S. L. Macaulay, M. A. Febbraio, and B. E. Kemp, “AMP-activated protein kinase—the fat controller of the energy railroad,” Canadian Journal of Physiology and Pharmacology, vol. 84, no. 7, pp. 655–665, 2006. View at Publisher · View at Google Scholar · View at Scopus
  19. W. W. Winder, H. A. Wilson, D. G. Hardie et al., “Phosphorylation of rat muscle acetyl-CoA carboxylase by AMP-activated protein kinase and protein kinase A,” Journal of Applied Physiology, vol. 82, no. 1, pp. 219–225, 1997. View at Publisher · View at Google Scholar · View at Scopus
  20. J. Park, Y.-D. Jeon, H.-L. Kim et al., “Interaction of Veratrum nigrum with Panax ginseng against obesity: a sang-ban relationship,” Evidence-Based Complementary and Alternative Medicine, vol. 2013, Article ID 732126, 13 pages, 2013. View at Publisher · View at Google Scholar · View at Scopus
  21. H. Wang, S.-Y. Li, C.-K. Zhao, and X. Zeng, “A system for screening agonists targeting β2- adrenoceptor from Chinese medicinal herbs,” Journal of Zhejiang University: Science B, vol. 10, no. 4, pp. 243–250, 2009. View at Publisher · View at Google Scholar · View at Scopus
  22. Z.-Z. Wang, W.-J. Zhao, X.-S. Zhang et al., “Protection of Veratrum nigrum L. var. ussuriense Nakai alkaloids against ischemia-reperfusion injury of the rat liver,” World Journal of Gastroenterology, vol. 13, no. 4, pp. 564–571, 2007. View at Publisher · View at Google Scholar · View at Scopus
  23. K.-S. Kim, H. J. Yang, E.-K. Choi et al., “The effects of complex herbal medicine composed of Cornus fructus, Dioscoreae rhizoma, Aurantii fructus, and Mori folium in obese type-2 diabetes mice model,” Oriental Pharmacy and Experimental Medicine, vol. 13, no. 1, pp. 69–75, 2013. View at Publisher · View at Google Scholar · View at Scopus
  24. H.-L. Kim, Y.-D. Jeon, J. Park et al., “Corni fructus containing formulation attenuates weight gain in mice with diet-induced obesity and regulates adipogenesis through AMPK,” Evidence-Based Complementary and Alternative Medicine, vol. 2013, Article ID 423741, 11 pages, 2013. View at Publisher · View at Google Scholar · View at Scopus
  25. M.-Y. Jeong, H.-L. Kim, J. Park et al., “Rubi Fructus (Rubus coreanus) activates the expression of thermogenic genes in vivo and in vitro,” International Journal of Obesity, vol. 39, no. 3, pp. 456–464, 2015. View at Publisher · View at Google Scholar · View at Scopus
  26. J. L. Ramírez-Zacarías, F. Castro-Muñozledo, and W. Kuri-Harcuch, “Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with oil red O,” Histochemistry, vol. 97, no. 6, pp. 493–497, 1992. View at Publisher · View at Google Scholar · View at Scopus
  27. M. M. Bradford, “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding,” Analytical Biochemistry, vol. 72, no. 1-2, pp. 248–254, 1976. View at Publisher · View at Google Scholar · View at Scopus
  28. N.-H. Kim, S.-K. Choi, S.-J. Kim et al., “Green tea seed oil reduces weight gain in C57BL/6J mice and influences adipocyte differentiation by suppressing peroxisome proliferator-activated receptor-γ,” Pflugers Archiv, vol. 457, no. 2, pp. 293–302, 2008. View at Publisher · View at Google Scholar · View at Scopus
  29. Z. Wu, Y. Xie, N. L. R. Bucher, and S. R. Farmer, “Conditional ectopic expression of C/EBPβ in NIH-3T3 cells induces PPARγ and stimulates adipogenesis,” Genes and Development, vol. 9, no. 19, pp. 2350–2363, 1995. View at Publisher · View at Google Scholar · View at Scopus
  30. Y. Xing, F. Yan, Y. Liu, Y. Liu, and Y. Zhao, “Matrine inhibits 3T3-L1 preadipocyte differentiation associated with suppression of ERK1/2 phosphorylation,” Biochemical and Biophysical Research Communications, vol. 396, no. 3, pp. 691–695, 2010. View at Publisher · View at Google Scholar · View at Scopus
  31. A. Yao-Borengasser, N. Rasouli, V. Varma et al., “Lipin expression is attenuated in adipose tissue of insulin-resistant human subjects and increases with peroxisome proliferator-activated receptor γ activation,” Diabetes, vol. 55, no. 10, pp. 2811–2818, 2006. View at Publisher · View at Google Scholar · View at Scopus
  32. M. Fulco and V. Sartorelli, “Comparing and contrasting the roles of AMPK and SIRT1 in metabolic tissues,” Cell Cycle, vol. 7, no. 23, pp. 3669–3679, 2008. View at Publisher · View at Google Scholar · View at Scopus
  33. X. Liu, R. R. Chhipa, I. Nakano, and B. Dasgupta, “The AMPK inhibitor compound C is a potent AMPK-independent antiglioma agent,” Molecular Cancer Therapeutics, vol. 13, no. 3, pp. 596–605, 2014. View at Publisher · View at Google Scholar · View at Scopus
  34. B. M. Spiegelman and J. S. Flier, “Obesity and the regulation of energy balance,” Cell, vol. 104, no. 4, pp. 531–543, 2001. View at Publisher · View at Google Scholar · View at Scopus
  35. C. A. Luque and J. A. Rey, “Sibutramine: a serotonin-norepinephrine reuptake-inhibitor for the treatment of obesity,” Annals of Pharmacotherapy, vol. 33, no. 9, pp. 968–978, 1999. View at Publisher · View at Google Scholar · View at Scopus
  36. A. M. Heck, J. A. Yanovski, and K. A. Calis, “Orlistat, a new lipase inhibitor for the management of obesity,” Pharmacotherapy, vol. 20, no. 3, pp. 270–279, 2000. View at Publisher · View at Google Scholar · View at Scopus
  37. J. Heo, Dongeuibogam(1613), Dongeuibogam Publishing Company, Seoul, Republic of Korea, 1st edition, 2006.
  38. D. Bensky, S. Clavey, and E. Stõger, Chinese Herbal Medicine: Materia Medica, Eastland Press, Seattle, Wash, USA, 3rd edition, 2004.
  39. W. A. Jacobs and Y. Sato, “The veratrine alkaloids; the structure of jervine,” The Journal of Biological Chemistry, vol. 175, no. 1, pp. 57–65, 1948. View at Google Scholar
  40. J. Tang, H.-L. Li, Y.-H. Shen et al., “Antitumor activity of extracts and compounds from the rhizomes of Veratrum dahuricum,” Phytotherapy Research, vol. 22, no. 8, pp. 1093–1096, 2008. View at Publisher · View at Google Scholar · View at Scopus
  41. J. Tang, H.-L. Li, Y.-H. Shen et al., “Antitumor and antiplatelet activity of alkaloids from Veratrum dahuricum,” Phytotherapy Research, vol. 24, no. 6, pp. 821–826, 2010. View at Publisher · View at Google Scholar · View at Scopus
  42. K. C. Huang, The Pharmacology of Chinese Herbs, Second Edition, Taylor & Francis, New York, NY, USA, 2nd edition, 1998. View at Publisher · View at Google Scholar
  43. J. J. Díez and P. Iglesias, “The role of the novel adipocyte-derived protein adiponectin in human disease: an update,” Mini-Reviews in Medicinal Chemistry, vol. 10, no. 9, pp. 856–869, 2010. View at Publisher · View at Google Scholar · View at Scopus
  44. N. B. Ruderman, D. Carling, M. Prentki, and J. M. Cacicedo, “AMPK, insulin resistance, and the metabolic syndrome,” The Journal of Clinical Investigation, vol. 123, no. 7, pp. 2764–2772, 2013. View at Publisher · View at Google Scholar · View at Scopus
  45. B. Huang, H. D. Yuan, D. Y. Kim, H. Y. Quan, and S. H. Chung, “Cinnamaldehyde prevents adipocyte differentiation and adipogenesis via regulation of peroxisome proliferator-activated receptor-γ (PPARγ) and AMP-activated protein kinase (AMPK) pathways,” Journal of Agricultural and Food Chemistry, vol. 59, no. 8, pp. 3666–3673, 2011. View at Publisher · View at Google Scholar · View at Scopus
  46. H. Kim and S.-Y. Choung, “Anti-obesity effects of Boussingaulti gracilis miers var. pseudobaselloides bailey via activation of AMP-activated protein kinase in 3T3-L1 cells,” Journal of Medicinal Food, vol. 15, no. 9, pp. 811–817, 2012. View at Publisher · View at Google Scholar · View at Scopus
  47. N. B. Ruderman, X. J. Xu, L. Nelson et al., “AMPK and SIRT1: a long-standing partnership?” American Journal of Physiology, Endocrinology and Metabolism, vol. 298, no. 4, pp. E751–E760, 2010. View at Publisher · View at Google Scholar · View at Scopus
  48. E. S. Buhl, N. Jessen, R. Pold et al., “Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying features of the insulin resistance syndrome,” Diabetes, vol. 51, no. 7, pp. 2199–2206, 2002. View at Publisher · View at Google Scholar · View at Scopus
  49. X. M. Song, M. Fiedler, D. Galuska et al., “5-Aminoimidazole-4-carboxamide ribonucleoside treatment improves glucose homeostasis in insulin-resistant diabetic (ob/ob) mice,” Diabetologia, vol. 45, no. 1, pp. 56–65, 2002. View at Publisher · View at Google Scholar · View at Scopus
  50. X. Hou, S. Xu, K. A. Maitland-Toolan et al., “SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase,” The Journal of Biological Chemistry, vol. 283, no. 29, pp. 20015–20026, 2008. View at Publisher · View at Google Scholar · View at Scopus
  51. N. L. Price, A. P. Gomes, A. J. Y. Ling et al., “SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function,” Cell Metabolism, vol. 15, no. 5, pp. 675–690, 2012. View at Publisher · View at Google Scholar · View at Scopus
  52. C. Cantó, Z. Gerhart-Hines, J. N. Feige et al., “AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity,” Nature, vol. 458, no. 7241, pp. 1056–1060, 2009. View at Publisher · View at Google Scholar · View at Scopus
  53. A. Hemminki, D. Markie, I. Tomlinson et al., “A serine/threonine kinase gene defective in Peutz-Jeghers syndrome,” Nature, vol. 391, no. 6663, pp. 184–187, 1998. View at Publisher · View at Google Scholar · View at Scopus
  54. G. J. Gowans, S. A. Hawley, F. A. Ross, and D. G. Hardie, “AMP is a true physiological regulator of AMP-activated protein kinase by both allosteric activation and enhancing net phosphorylation,” Cell Metabolism, vol. 18, no. 4, pp. 556–566, 2013. View at Publisher · View at Google Scholar · View at Scopus
  55. L. A. Witters, T. D. Watts, D. L. Daniels, and J. L. Evans, “Insulin stimulates the dephosphorylation and activation of acetyl-CoA carboxylase,” Proceedings of the National Academy of Sciences of the United States of America, vol. 85, no. 15, pp. 5473–5477, 1988. View at Publisher · View at Google Scholar · View at Scopus
  56. D. Carling, V. A. Zammit, and D. G. Hardie, “A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis,” FEBS Letters, vol. 223, no. 2, pp. 217–222, 1987. View at Publisher · View at Google Scholar · View at Scopus
  57. L. Luo, S. Jiang, D. Huang, N. Lu, and Z. Luo, “MLK3 phophorylates AMPK independently of LKB1,” PLoS ONE, vol. 10, no. 4, Article ID e0123927, 2015. View at Publisher · View at Google Scholar · View at Scopus
  58. B. N. M. Zordoky, J. Nagendran, T. Pulinilkunnil et al., “AMPK-dependent inhibitory phosphorylation of ACC is not essential for maintaining myocardial fatty acid oxidation,” Circulation Research, vol. 115, no. 5, pp. 518–524, 2014. View at Publisher · View at Google Scholar · View at Scopus